A significant gap in the field of cancer research is the lack of unbiased, genome-wide studies describing how activated Notch regulates the genomes of cancer cells. We propose to begin to fill this gap by elucidating how activated Notchl (ICNI) interacts with the genomes of T-ALL cells to support leukemia cell growth. We will address this central issue in T-ALL pathogenesis by pursuing the following two specific aims: Aim 1. To determine how Notchl transactivates target genes Current models for Notch regulation of target genes have been based on analyses of only a few genes (e.g., i-ies1) in diverse cellular contexts. Our preliminary data suggest that individual Notchl binding sites show differential sensitivity to y-secretase inhibitors (GSls), a characteristic that may define dynamic NREs and which points to an unexpected pool of GSI-refractory chromatin-associated Notchl. In this aim, we will first use ChlP-Seq to determine the GSI-sensitivity of chromatin-associated Notchl on a genome-wide basis. Then, with a full set of robust target genes and GSI-sensitive Notchl binding sites in hand, we will test and refine models of Notchl regulation of gene expression in T-ALL cells. We anticipate that these studies will illuminate fundamental mechanisms of target gene regulation by Notch and reveal new opportunities for therapeutic targeting ofthe Notch pathway. Aim 2. To determine the contributions of cis-regulatory co-factors to Notchl target gene expression Motif analysis carried out in human and murine T-ALL cells showed that Notchl genomic binding sites are often flanked by motifs for Runx factors or Ets factors, or overlap precisely with binding sites for Znf143. In preliminary data, we have observed that Runx factors and Ets factors appear to co-regulate important Notchl target genes such as iL7R that contribute to the growth and survival of T-ALL cells. We will expand upon these data to study how Ets and Runx factors co-regulate key target genes, and will also elucidate the pervasive, but complex relationship of Notchl and Znf143. Together, the work described in these two aims will substantially advance our understanding of the molecular pathogenesis of T-ALL, and in doing so create new opportunities for targeting of the Notch pathway in cancer and in other forms of disease related to dysregulated Notch signaling.